Recent advances in the field of single molecule spectroscopy have enabled multiplexed, ultra-sensitive, and highly specific detection and quantification of biomolecules. Despite their many advantages over ensemble-averaged detection methods, most single molecule spectroscopies remain tools which are used exclusively by highly trained researchers due to the expense and expertise required to build such setups. This proposal seeks to expand the user-base of single molecule spectroscopies by creating a simple, cost-effective, and ultra-stable commercial single molecule spectroscopic reader for diagnostic applications which can be operated by personnel with little to no experience in optics. The device will find numerous uses in hospitals and diagnostic labs as well as in biomedical research. It will help guide physicians in early disease detection, assessment of health status, and initiation of treatment, while it concomitantly allows monitoring the public and environment for disease outbreaks, drug resistance trends, and potential bioterrorism attacks. The envisioned device couples the single-well multiplexing capabilities of Alternating Laser Excitation (ALEX) -a single molecule spectroscopy pioneered by Prof. Shimon Weiss at UCLA- with the exquisite sensitivity of confocal fluorescence detection. Nesher Technologies, Inc. (NTI) has been established to commercialize this enabling technology and through this proposal intends to develop a prototype table-top single molecule reader with improved multiplexing capabilities by expanding the currently existing three-color ALEX technology to four colors (4c-ALEX). With this reader as its foundation, NTI's long-term goal is to develop a variety of simple "mix-and-read" nucleic acid- and protein-based tests which can simultaneously detect and accurately quantify multiple genetic aberrations, biomarkers, and/or pathogens, in a single patient (or environmental) sample. Reagent development and proof-of-concept testing is currently ongoing and funded through several grants. The Specific Aims of the present proposal are: 1). NTI will build a compact, low-cost ($40,000-$45,000), and ultra-stable 4c-ALEX single molecule reader (suitable for prototype development in Phase II). 2). The device's single-well multiplexing capabilities will be demonstrated by the simultaneous detection and quantification of multiple targets in a single sample using dye-labeled nucleic acid and protein reporter molecules. 3). The device's stability and accuracy will be demonstrated by comparing its performance to an existing non- commercial 3c-ALEX single molecule setup in Prof. Weiss'lab. 4). Existing data acquisition and analysis software will be streamlined to minimize user input and fully automate the data-acquisition/analysis process for 4c-ALEX. PUBLIC HEALTH RELEVANCE: Development of a highly multiplexed, ultrasensitive and -specific, quantitative, low-cost automated medical diagnostic testing system, capable of quickly identifying specific healthcare-associated targets among the multitude of possibilities from a single patient sample, radically pushes the limits of current technologies. With this grant application Nesher Technologies, Inc. proposes a first major step towards prototype development of a fully integrated, small-footprint, very innovative spectroscopic biomolecule reader suitable for commercialization. It will complement Nesher Technologies'ongoing, federally-funded efforts of reagent development for highly multiplexed, ultrasensitive and -specific, quantitative, fully automated, and cost-effective tests for early cancer, genetic diseases, and bioterror agent detection, thereby translating cutting-edge innovations in nanotechnology into benefits for the society at large by saving human lives and reducing healthcare costs.